Method for use of homopiperazinium compounds in the treatment of cancer

ABSTRACT

A method for treating cancer comprising administering to a patient in need thereof an effective amount of a compound having the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1  and R 2  are independently alkyl of 1 to 6 carbon atoms or cycloalkyl of 3 to 6 carbon atoms, Xa is CH or N, Ya is hydrogen or a substituent, each of which is independently selected; n is an integer from 1 to 5, J is a counter ion.

FIELD OF THE DISCLOSURE

The present disclosure relates to novel use and method for the treatmentor prevention for treating or preventing cancer or for reducing orstopping the proliferation of cancer cells.

BACKGROUND OF THE DISCLOSURE

A broad group of diseases involving unregulated cell growth is known ascancer or as malignant neoplasia. In cancer, cells divide and growuncontrollably, causing the cells to form lumps or tumors. The cancermay also spread to more distant parts of the body through the lymphaticsystem or bloodstream. Not all tumors are cancerous; benign tumors donot invade neighboring tissues and do not spread throughout the body.

For the treatment of cancer, chemotherapeutic, immunotherapeutic orimmunomodulatory and antiangiogenic agents have been reported. Agentscan be used as monotherapy (treatment with one agent) or as combinationtherapy (simultaneous, separate or sequential treatment with anotheragent). The treatments may also be combined with radiotherapy.

In this respect, a chemotherapeutic agent means a naturally occurring,semi-synthetic or synthetic chemical compound which, alone or viafurther activation, for example with radiations in the case ofradio-immunotherapy, inhibits or kills growing cells, and which can beused or is approved for use in the treatment of diseases of oncologicalnature, which are commonly also denominated as cancers. In theliterature, these agents are generally classified according to theirmechanism of action. In this matter, reference can be made, for example,to the classification made in “Cancer Chemotherapeutic Agents”, AmericanChemical Society, 1995, W. O. Foye Ed.

Even if several therapeutic agents have already been investigated andused, there is still a need for new and efficient therapeutic agents forthe treatment of cancer diseases.

One object of the present invention is to provide a method for thetreatment of various cancer diseases.

SUMMARY

In one aspect, there is provided a method, composition or use fortreating or preventing cancer, comprising administering an effectiveamount of a compound having the formula:

In one aspect, there is provided a combination for treating orpreventing cancer comprising a compound as defined herein and anadditional anticancer drug.

In one aspect, there is provided a method, composition or use forreducing or stopping the proliferation of cancer cells.

In one aspect, there is provided a combination for reducing or stoppingthe proliferation of cancer cells and an additional drug useful forreducing or stopping the proliferation of cancer cells.

In one aspect, there is provided a pharmaceutical composition comprisingan effective amount of a compound as defined herein and apharmaceutically acceptable carrier or excipient.

In one aspect, there is provided a pharmaceutical composition comprisingan effective amount of a compound as defined herein and optionally oneor more agents for treating or preventing cancer or for reducing orstopping the proliferation of cancer cells.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 4 show compound-induced dose-related inhibition of A549, H520and H82 cell proliferation;

FIGS. 5a and 5b illustrate the observed results for the MDA-MB-231mammary/breast gland adenocarcinoma assay;

FIGS. 6a, 6b, 7a and 7b are illustrating the antitumor effect of acompound of the invention in A549 tumor cell-bearing mice after 51 days;

FIGS. 8a, and 8b illustrate the in vitro cytotoxicity potentiation ofcisplatin and taxol in combination with a compound of the invention inA549 human lung adenocarcinoma;

FIGS. 9a and 9b illustrate the in vitro cytotoxicity potentiation ofcisplatin or and taxol in combination with a compound of the inventionin MCF7 human breast adenocarcinoma;

FIG. 10A: represents the tumor volume in nude mice treated with eithercisplatin, taxol or a compound of the invention; and

FIG. 10B represents the tumor weight in nude mice treated with eithercisplatin, taxol or a compound of the invention.

DESCRIPTION OF THE EMBODIMENTS

In one embodiment, Xa is CH. In one embodiment, Xa is N

In one embodiment, Ya is hydrogen or independently selected fromhalogen, amino, amidino, amido, azido, cyano, guanido, hydroxyl, nitro,nitroso, urea, sulfate, sulfite, sulfonate, sulphonamide, phosphate,phosphonate, acyl, acyloxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to6 carbon atoms, alkylthio of 1 to 6 carbon atoms, alkylamino of 1 to 6carbon atoms, alkanol of 1 to 6 carbon atoms, aralkyl, aryl of 6 to 10carbon atoms and 3 to 10 membered heterocycle.

In one embodiment, Ya is hydrogen or independently selected fromhalogen, amino, amidino, cyano, hydroxyl, nitro, urea, sulfate, sulfite,sulfonate, phosphate, phosphonate, acyl, acyloxy, alkyl of 1 to 6 carbonatoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms,alkylamino of 1 to 6 carbon atoms, and alkanol of 1 to 6 carbon atoms;aryl and heteroaryl.

In one embodiment, Ya is hydrogen or independently selected fromhalogen, cyano, hydroxyl, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6carbon atoms, heteroaryl of 6 members and aryl. In one embodiment, Ya ishydrogen or independently selected from halogen, cyano, hydroxyl, alkylof 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms and heteroaryl of6 members and aryl of 6 or 10 carbon atoms.

In one embodiment, the homopiperazinium compound is a compound whereinJ⁻ is a halogen, a sulphate, acetate or a sulfonate. In one embodiment,the homopiperazinium compound is a compound wherein J⁻ is a halogen or asulfonate.

In one embodiment, the homopiperazinium compound is a compound whereinJ⁻ is a halogen. In one embodiment, the halogen is iodide, chloride orbromide. In one embodiment, the halogen is iodide. In one embodiment,the halogen is chloride. In one embodiment, the halogen is bromide.

In one embodiment, the homopiperazinium compound is a compound whereinJ⁻ is a sulfonate. In one embodiment, the sulfonate is4-toluenesulfonate, phenylsulfonate or methanesulfonate. In oneembodiment, the sulfonate is 4-toluenesulfonate. In one embodiment, thesulfonate is phenylsulfonate. In one embodiment, the sulfonate ismethanesulfonate.

In one embodiment, n is 1 to 4. In one embodiment, n is 1 to 3. In oneembodiment, n is 1 or 2.

In one embodiment, R₁ and R₂ are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is CH;

Ya is hydrogen.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is N or CH;

Ya is hydrogen or independently selected from halogen, cyano, hydroxyl,alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, heteroarylof 6 members and aryl;

-   -   n is 1 or 2;    -   wherein J⁻ is fluoride, chloride, bromide, iodide, acetate,        sulfate or sulfonate such as tosylate, mesylate, besylate.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is N or CH;

Ya is hydrogen or independently selected from halogen, cyano, hydroxyl,alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, heteroarylof 6 members and aryl;

n is 1;

wherein J⁻ is fluoride, chloride, bromide, iodide, acetate, sulfate orsulfonate such as tosylate, mesylate, besylate.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is N;

Ya is hydrogen or independently selected from halogen, cyano, hydroxyl,alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, heteroarylof 6 members and aryl;

n is 1;

wherein J⁻ is fluoride, chloride, bromide, iodide, acetate, sulfate orsulfonate such as tosylate, mesylate, besylate.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is CH;

Ya is hydrogen or independently selected from halogen, cyano, hydroxyl,alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, heteroarylof 6 members and aryl;

n is 1;

wherein J⁻ is fluoride, chloride, bromide, iodide, acetate, sulfate orsulfonate such as tosylate, mesylate, besylate.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is CH;

Ya is hydrogen

n is 1;

wherein J⁻ is fluoride, chloride, bromide, iodide, sulfate or sulfonate.

In one embodiment, R1 and R2 are independently selected from methyl,ethyl, n-propyl, or i-propyl;

Xa is CH;

Ya is hydrogen

n is 1

wherein J⁻ is sulfonate.

In one embodiment, R1 and R2 are ethyl;

Xa is CH;

Ya is hydrogen

n is 1

wherein J⁻ is tosylate.

The term “alkyl” represents a linear or branched hydrocarbon moietyhaving 1 to 10 or preferably 1 to 6 carbon atoms, which may have one ormore unsaturation in the chain, and is optionally substituted. The term“lower alkyl” is also meant to include alkyls in which one or morehydrogen atom is replaced by a halogen, ie. an alkylhalide.

The term “cycloalkyl” represents an alkyl chain of 3 to 6 carbon atoms.

The term “alkoxy” represents an alkyl which is covalently bonded to theadjacent atom through an oxygen atom.

The term “alkylthio” represents an alkyl which is covalently bonded tothe adjacent atom through a sulfur atom.

The term “alkylamino” represents an alkyl which is covalently bonded tothe adjacent atom through a nitrogen atom and may be monoalkylamino ordialkylamino, wherein the alkyl groups may be the same or different.

The term “alkanol” represents an “alkyl” moiety for which one of thehydrogens has been replaced by an hydroxyl group.

The term “aralkyl” represents an aryl group attached to the adjacentatom by a C₁₋₆ alkyl.

The term “aryl” represents a carbocyclic moiety containing at least onebenzenoid-type ring (i.e. may be monocyclic or polycyclic) having 6 to10 carbon atoms, and which may be optionally substituted with one ormore substituents. Alternatively, the ring may be containing 6 carbonatoms.

The term “acyl” is defined as a radical derived from a carboxylic acid,obtained by replacement of the —OH group. Like the acid to which it isrelated, an acyl radical may be derived form a straight chain, branchedchain or cyclic alkyl or aryl.

The term “acyloxy” represents an acyl which is covalently bonded to theadjacent atom through an oxygen atom.

The term “halogen atom” is specifically a fluoride atom, chloride atom,bromide atom or iodide atom.

The term “heterocycle” represents a 3 to 10 membered optionallysubstituted saturated, unsaturated cyclic moiety wherein said cyclicmoeity is interrupted by at least one heteroatom selected from oxygen(O), sulfur (S) or nitrogen (N). Alternatively, heterocycles may be 3 to6 membered ring or 5 to 6 membered ring. Heterocycles may be monocyclicor polycyclic rings.

The term “heteroaryl” represents an aryl ring wherein said ring isinterrupted by at least one heteroatom selected from oxygen (O), sulfur(S) or nitrogen (N). Preferably, heteroaryl rings may be containing 5 or6 ring members.

The term “counterion” is meant to include a pharmaceutically acceptableion that accompanies an ionic species (e.g. the homopiperazinium moiety)in order to maintain electric neutrality. Counterions can also beprovided by the conjugate bases derived from pharmaceutically acceptableinorganic and organic acids such as hydrochloric, hydrobromic,sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic,lactic, salicylic, succinic, paratoluene-sulphonic, tartaric, acetic,trifluoroacetic, citric, methanesulphonic, formic, benzoic, malonic,naphthalene 2 sulphonic and benzenesulphonic acids.

The term “independently” means that a substituent can be the same or adifferent definition for each item.

As used herein, “treatment” or “treating” refers to at least i)controlling or ameliorating at least one disease described herein, atleast for the duration of said treatment.

Although not limited to such patients, “prevention” or “prophylaxis”treatment (which may be used interchangeably) is expected to beparticularly useful to the treatment of patients who have suffered aprevious episode associated with diseases described herein, or areotherwise considered to be at increased risk of said diseases. Asuccessful preventive treatment would normally be expected to i) reducethe occurrences of a further episode, ii) reduce its severity or iii)prevent occurrences of further episodes, at least for the duration ofthe therapy.

In one embodiment, the present disclosure provides a method, use orcomposition for treating cancer comprising administering an effectiveamount of at least one compound as defined herein.

In one embodiment, the present disclosure provides a method, use orcomposition for limiting, or inhibiting the proliferation of cancercells, or for causing death of cancer cells in a patient, comprisingadministering an effective amount of at least one compound as definedherein.

In another embodiment, the expression “cancer” includes, but is notlimited to carcinomas, sarcomas, melanomas; lymphoma, leukemia andmyelomas; blastomas; germ cell tumor; glioma and other CNS cancers.

In one embodiment, the carcinoma is a cancer of the bladder, breast,cervix, colon, esophagus, kidney, liver, larynx, lung (small andnon-small cell lung cancer), oral cavity, ovary, pancreas, pleura,prostate, skin (basal and squamous), stomach, thyroid or uterus.

In one embodiment, the sarcoma is osteosarcoma, chondrosarcoma,liposarcoma, neurosarcoma, rhabdomyosarcoma, Erwing sarcoma orfibrosarcoma.

In one embodiment, the melanoma is malignant melanoma, lentigo malignamelanoma, superficial spreading melanoma, acral lentiginous melanoma,mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplasticmelanoma, amelanotic melanoma or soft-tissue melanoma.

In one embodiment, the lymphoma, leukemia and myelomas is acutelymphocytic leukemia, B-cell lymphoma, Burketts lymphoma, Hodgkin andNon-Hodgkin lymphoma, acute and chronic myelogenous leukemias,promyelocytic leukemia or multiple myeloma.

In one embodiment, the blastoma is a blastoma derived from immature“precursor” cells or embryonic tissue, neuroblastoma, retinoblastoma,pleuropulmonary blastoma, nephroblastoma (Wilms tumor) orhepatoblastoma.

In one embodiment, the germ cell tumor is a seminoma, dysgerminoma orteratocarcinoma tumor.

In one embodiment, the glioma and other CNS cancers are ependymomas,astrocytomas, oligodendrogliomas, glioblastomas or oligoastrocytomas.

In a further embodiment the invention relates to a method, use orcomposition for the treatment of cell proliferation, migration orapoptosis of cancer cells, or angiogenesis, in a human or non-humanmammalian body.

In a further embodiment the invention relates to a method, use orcomposition for reducing or stopping the proliferation of cancer cells.By proliferation it is meant cell proliferation resulting fromunregulated and/or undesirable cell growth, for example caused byexcessive cell division, cell division at an accelerated rate and/orundesirable cell survival.

In another embodiment, the present disclosure provides a combinationcomprising a therapeutically effective amount of a compound, as definedherein, and a therapeutically effective amount of at least one or moretherapeutic agents useful in the method of the present disclosureselected from: Alkylating agents, Anti-metabolites, Plant alkaloids andterpenoids, Vinca alkaloids, Podophyllotoxin, Taxanes, Topoisomeraseinhibitors, and Cytotoxic antibiotics

In another embodiment, the present invention provides a combinationcomprising a therapeutically effective amount of a compound, as definedherein, and a therapeutically effective amount of at least one or moretherapeutic agents useful in the method of the present disclosureincluding but not limited to imatinib, paclitaxel, docetaxel, cisplatin,doxorubicine, vinblastine, zoledronate and/or in conjunction withantimetastatic agents, antiangionevic agents such as avastatin, andantiapoptotic compounds such as Velcade™, agents targeting synthesis ofestrogens or estrogen signaling through estrogen receptors including butnot limited to arimidex and tamoxifen, agents targeting biosynthesis ofandrogens or androgen signaling through the androgen receptor includingbut not limited to bicalutamide, agents targeting HER2 including but notlimited to trastuzumab, agents targeting BRAF including but not limitedto Vemurafenib, or agents targeting members of the MAP kinase family ortheir upstream or downstream effector kinases.

It will be clear to a person of ordinary skill that the amounts and/orratios of therapeutic agents will be readily adjusted. It will beunderstood that the scope of combinations described herein is notparticularly limited, but includes in principle any therapeutic agentuseful for preventing or treating the diseases described herein.

It will also be appreciated that the amounts and/or ratios oftherapeutic agents for use in treatment will vary not only with theparticular agent selected but also with the route of administration, thenature of the condition for which treatment is required and the age andcondition of the patient and will be ultimately at the discretion of theattendant physician.

The homopiperazinium compounds defined herein can be administeredconcurrently to the one or more agents used herein in the methods andcombinations. The desired doses may conveniently be presented in asingle dose or as divided dose administered at appropriate intervals,for example as two, three, four or more doses per day or continuouslysuch as in a perfusion. The compound can be administered on a dosageregimen distinct to the one or more agents used herein in the methodsand combinations. Alternatively, the compound can be administeredsequentially or concurrently in distinct formulations or in a commonformulation.

Pharmaceutical compositions may comprise pharmaceutically acceptablecarriers.

The formulations may, where appropriate, be conveniently presented indiscrete dosage units and may be prepared by any of the methods wellknown in the art of pharmacy. All methods include the step of bringinginto association the active compound with a liquid carrier or solidcarrier or both and then, if necessary, shaping the product into thedesired formulation.

Pharmaceutical compositions suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may be in theform of, for example, aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The compounds and combinations according to the invention may also beformulated for parenteral administration (e.g. by injection, for examplebolus injection or continuous infusion) and may be presented in unitdose form in ampoules, pre-filled syringes, small volume infusion or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

The following examples are provided to further illustrate details forthe preparation and use of the compounds of the present invention. Theyare not intended to be limitations on the scope of the instant inventionin any way, and they should not be so construed. Furthermore, thecompounds described in the following examples are not to be construed asforming the only genus that is considered as the invention, and anycombination of the compounds or their moieties may itself form a genus.

General Experimental Methods

Reactions were performed under argon atmosphere. Melting points areuncorrected. ¹H NMR spectra were recorded at 400 MHz and were referencedto the peak for residual solvent. ¹³C NMR spectra were recorded at 100MHz (¹³C NMR at 75 MHz for Preparative Example) and were referenced tothe peak for residual solvent. Chemical shifts in ¹H and ¹³C NMR spectraare reported in ppm. All reagents (e.g. 1-methylhomopiperazine andhomopiperazine) can be obtained commercially, e.g. from Sigma-AldrichCo. Usual solvents and chemicals can be obtained commercially, e.g. fromVWR, A&C or Fisher and were also “reagent” grade. Chromatography wasperformed using Silica Gel 60 (Merck; 230-400 mesh). Accurate massmeasurements were performed on a LC-MSD-Tof instrument from Agilenttechnologies in positive electrospray mode. Protonated molecular ions(M+H)⁺ was used for empirical formula confirmation.

Preparative Example 1: 1-Phenyl-4-ethyl-homopiperazine RP-HPLCConditions (Preparative Example)

HPLC analysis were performed on a Waters C18 reversed-phase analyticalcolumn (5 μm, Atlantis, 100×3.9 mm) using a flow rate of 1 mL/min and agradient of 0% to 95% A/B over 15 min, where A=0.1% aqueous Formic Acidand B=CH₃CN+0.1% FA

A solution of homopiperazine (50 g, 499.1 mmol, 1.2 eq), iodobenzene(84.86 g, 416 mmol, 1 eq), ethylene glycol (46.4 mL, 832 mmol, 2 eq),CuI (3.96 g, 20.8 mmol, 5% mol) and K₃PO₄ (88.3 g, 416 mmol, 1 eq) andisopropanol (416 mL) was stirred at reflux for 46 h. The resultingmixture was cooled down to room temperature and isopropanol wasevaporated. Water (200 mL), containing NH₄OH (1%), and EtOAc (250 mL)were added to the mixture. The aqueous layer was extracted with EtOAc(4×200 mL), and the combined organic layers were washed with brine(2×200 mL), dried over Na₂SO₄, filtered and evaporated. The crudeproduct (52.2 g) was obtained as a brown oil and was used in the nextstep without any purification.

To a solution of 1-phenylhomopiperazine (52.2 g, 292.6 mmol, 1 eq) indichloromethane (300 mL) were added at 0° C. Et₃N (90 mL, 890 mmol, 3eq) and Ac₂O (112.15 mL, 1186 mmol, 4 eq). The mixture was stirred atroom temperature for 2 h. A 4N NaOH solution (200 mL) was added and theresulting mixture was extracted with CH₂Cl₂ (3×150 mL). The combinedorganic layers were washed with brine (2×150 mL), dried over Na₂SO₄ andevaporated. The resulting oil was coevaporated with EtOH (3×), EtOAc(3×) and Et₂O (3×), to give 68.03 g of crude product which was used inthe next step without any purification.

To a suspension of AlLiH₄ (28.46 g, 750 mmol, 1.5 eq) in THF (400 mL) at0° C. was added dropwise a solution of 1-phenyl-4-acyl-homopiperazine(109 g, 500 mmol, 1 eq) in THF (500 mL). The mixture was warmed up toroom temperature and stirred for 24 h. The mixture was then cooled downto 0° C. and H₂O (350 mL) was added dropwise. THF was evaporated, TBME(400 mL) was added and the mixture was filtered on Celite®. The layerswere separated, and the aqueous phase was extracted with TBME (3×150mL). The organic layers were combined and washed with brine (2×150 mL),dried over Na₂SO₄, filtered and evaporated. The crude product waspurified by chromatography on silica gel using 100% hexanes and agradient of 0% to 20% MeOH in CH₂Cl₂. The desired compound1-Phenyl-4-ethyl-homopiperazine was obtained as a orange oil (38.1 g,19% overall yield): ¹H NMR CDCl₃ (ppm): 7.26 (dd, 2H), 6.73 (m, 3H),3.61 (t, 2H), 3.53 (t, 2H), 2.81 (m, 2H), 2.62 (m, 4H), 2.03 (m, 2H),1.12 (t, 3H); ¹³C NMR CDCl₃ (ppm): 148.8, 128.9, 115.4, 111.2, 54.7,53.8, 51.3, 48.3, 47.6, 27.4, 12.1.

Preparative Example 2: 1-methyl-4-phenylhomopiperazine

In a flame-dried round bottom flask under nitrogen, iodobenzene (1 eq,1.47 mmol), N-methylhomopiperazine (1.2 eq, 1.76 mmol), ethylene glycol(2 eq, 2.94 mmol), CuI (5% mol) and K3PO4 (2 eq, 2.94 mmol) weresuspended in isopropanol (3 ml). The mixture was refluxed with stirringfor 17 hours. The resulting mixture was cooled down to room temperatureand water was added (5 ml). The mixture was extracted with ether (4×10ml) and the combined organic extracts washed with brine, dried overNa2SO4 and evaporated to dryness under vacuum. The crude product waspurified using silica gel flash chromatography using a gradient of 0% a7.5% (2M NH3)MeOH in chloroform. The desired product was obtained as ayellow oil. (yield 64%).

Preparative Example 3: 4-Methyl-1-(4-methoxyphenyl)-homopiperazine

To a solution of 1-methylhomopiperazine (918 mg, 8.04 mmol) in i-PrOH(10 mL) and ethylene glycol (0.90 mL) was added CuI (76 mg, 0.4 mmol),K₃PO₄ (1.706 g, 8.04 mmol) and 1-iodoanisole (1.882 g, 8.04 mmol). Thereaction mixture was stirred under reflux for 48 h. After cooling, themixture was taken in EtOAc and 0.5% aqueous NH₄OH. The layers wereseparated and the aqueous layer was extracted 3 times with EtOAc.

The organic layers were combined, washed with brine, dried (Na₂SO₄) andconcentrated to a residue that was purified by column chromatography onsilica gel (5/95/0 to 19.9/80/0.1 MeOH/DCM/NH₄OH). Evaporation of thecollected fractions yielded the title compound as a beige solid (711 mg,40% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.83 (d, 2H), 6.64 (d, 2H), 3.74(s, 3H), 3.53 (t, 2H), 3.43 (t, 2H), 2.71 (t, 2H), 2.59 (t, 2H), 2.39(s, 3H), 2.01 (quad, 2H); HPLC: condition A, 4.67 min, >99% homogeneity;ES-MS [M+H⁺]=221.2.

Preparative Example 4: 4-Methyl-1-(2,4-dimethoxyphenyl)-homopiperazine

To a solution of 1-methylhomopiperazine (1.377 g, 11.98 mmol) in i-PrOH(15 mL) and ethylene glycol (1.35 mL) was added CuI (114 mg, 0.60 mmol),K₃PO₄ (2.540 g, 11.98 mmol) and 1-iodo-2,4-dimethoxybeznene (3.160 g,11.98 mmol). The reaction mixture was stirred under reflux for 7 days.After cooling, the mixture was taken in EtOAc and 0.5% aqueous NH₄OH.The layers were separated and the aqueous layer was extracted 3 timeswith EtOAc. The organic layers were combined, washed with brine, dried(Na₂SO₄) and concentrated to a residue that was purified by columnchromatography on silica gel (5/95/0 to 19.9/80/0.1 MeOH/DCM/NH₄OH).Evaporation of the collected fractions yielded the title compound as abrown oil (614 mg, 20% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.88 (d, 1H),6.45 (s, 1H), 6.38 (d, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.25 (m, 4H),2.75 (m, 4H), 2.40 (s, 3H), 1.96 (quad, 2H); HPLC: condition A, RT=4.80min, 95.3% homogeneity.

Preparative Example 5:4-Methyl-1-(4-trifluoromethylphenyl)-homopiperazine

To a solution of 1-methylhomopiperazine (1.193 g, 10.45 mmol) in i-PrOH(10 mL) and ethylene glycol (1.16 mL) was added CuI (100 mg, 0.52 mmol),K₃PO₄ (2.218 g, 10.45 mmol) and 4-iodobenzotrifluoride (2.842 g, 10.45mmol). The reaction mixture was stirred under reflux for 24 h. Aftercooling, the mixture was taken in EtOAc and 0.5% aqueous NH₄OH. Thelayers were separated and the aqueous layer was extracted 3 times withEtOAc. The organic layers were combined, washed with brine, dried(Na₂SO₄) and concentrated to a residue which was purified by columnchromatography on silica gel (2/95/0 to 9.9/90/0.1 MeOH/DCM/NH₄OH).Evaporation of the collected fractions yielded the title compound as acolorless oil (713 mg, 26% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d,2H), 6.68 (d, 2H), 3.60 (m, 2H), 3.51 (m, 2H), 2.71 (m, 2H), 2.56 (m,2H), 2.38 (s, 3H), 2.02 (quad, 2H); HPLC: condition A, RT=5.20 min,98.7% homogeneity.

Preparative Example 6: 4-Methyl-1-(4-trifluorobenzyl)-homopiperazine

At 100° C., 1-methylhomopiperazine (1.22 g, 10.68 mmol) was allowed toreact with 4-bromobenzotrifluoride (2 g, 8.9 mmol) using catalyticamount of Pd₂(dba)₃ (82 mg, 0.089 mmol) and BINAP (166 mg, 0.267 mmol),NaOtBu (1.2 g, 12.46 mmol) with without solvent (neat). The reactionmixture was stirred under reflux for 1 h15. After cooling, the mixturewas taken in DCM and H₂O. The layers were separated and the aqueouslayer was extracted 3 times with DCM. The organic layers were combined,dried (Na₂SO₄) and concentrated to a residue which was purified bycolumn chromatography on silica gel (4.9/95/0.1 MeOH/DCM/NH₄OH).Evaporation of the collected fractions yielded the title compound as abrown oil (1.64 g, 72% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, 2H),6.98 (d, 2H), 3.59 (t, 2H), 3.50 (t, 2H), 2.69 (m, 2H), 2.54 (m, 2H),2.38 (s, 3H), 2.01 (m, 2H); ¹³C NMR (CDCl₃) δ 27.1, 46.3, 47.7, 48.0,56.6, 57.3, 110.3, 116.2, 116.5, 116.8, 117.1, 123.5, 126.0, 126.1,126.2, 150.9; MS (+) 259.2; HPLC: condition A, RT=5.38 min, >98%homogeneity.

Preparative Example 7: 4-Methyl-1-(4-cyanophenyl)-homopiperazine

To a solution of 1-methylhomopiperazine (955 mg, 8.36 mmol) in THF (17mL) was added Pd₂(dba)₃ (957 mg, 1.05 mmol), BINAP (1.302 g, 2.09 mmol),NaOt-Bu (938 mg, 9.76 mmol) and 4-iodobenzonitrile (1.596 g, 6.97 mmol).The reaction mixture was stirred under reflux for 1 h. After cooling,the mixture was diluted with Et₂O (200 mL), filtered and evaporated. Thecrude product was purified by column chromatography on silica gel(0/95/0 to 14.9/85/0.1 MeOH/DCM/NH₄OH). Evaporation of the collectedfractions yielded the title compound as a beige solid (915 mg, 61%yield). ¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, 2H), 6.63 (d, 2H), 3.59 (t,2H), 3.49 (t, 2H), 2.69 (m, 2H), 2.55 (m, 2H), 2.37 (s, 3H), 2.00 (quad,2H); HPLC: condition A, RT=4.48 min, 95.0% homogeneity.

Preparative Example 8: 4-Methyl-1-(3-methylphenyl)-homopiperazine

At 100° C., 1-methylhomopiperazine (1.6 g, 14 mmol) was allowed to reactwith 3-bromotoluene (2 g, 11.7 mmol) using catalytic amount of Pd₂(dba)₃(107 mg, 0.1169 mmol) and BINAP (218 mg, 0.35 mmol), NaOtBu (1.6 g,16.37 mmol) with without solvent (neat). The reaction mixture wasstirred under reflux for 4 h. After cooling, the mixture was taken inDCM and H₂O. The layers were separated and the aqueous layer wasextracted 3 times with DCM. The organic layers were combined, dried(Na₂SO₄) and concentrated to a residue which was purified by columnchromatography on silica gel (4.9/95/0.1 MeOH/DCM/NH₄OH). Evaporation ofthe collected fractions yielded the title compound as a brown oil (1.28g, 54% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.16 (t, 1H), 6.56 (m, 3H),3.61 (m, 2H), 3.52 (m, 2H), 2.74 (m, 2H), 2.61 (m, 2H), 2.43 (s, 3H),2.38 (s, 3H), 2.06 (m, 2H); ¹³C NMR (CDCl₃) δ 21.1, 27.5, 46.3, 47.9,48.1, 56.8, 57.9, 108.4, 111.9, 116.4, 128.8, 138.5, 149.0; MS (+)205.2; HPLC: condition A, RT=4.87 min, 100% homogeneity.

Preparative Example 9: 4-Methyl-1-(3,5-dimethoxyphenyl)-homopiperazine

At 100° C., 1-methylhomopiperazine (1.36 g, 11.88 mmol) was allowed toreact with 1-bromo-3,5-dimethoxybenzene (2.15 g, 9.9 mmol) usingcatalytic amount of Pd₂(dba)₃ (90.65 mg, 0.099 mmol) and BINAP (185 mg,0.297 mmol), NaOtBu (1.34 g, 13.86 mmol) with without solvent (neat).The reaction mixture was stirred under reflux for 23 h. After cooling,the mixture was taken in DCM and H₂O. The layers were separated and theaqueous layer was extracted 3 times with DCM. The organic layers werecombined, dried (Na₂SO₄) and concentrated to a residue which waspurified by column chromatography on silica gel (4.9/95/0.1MeOH/DCM/NH₄OH). Evaporation of the collected fractions yielded thetitle compound as a brown oil (1.04 g, 46% yield). ¹H NMR (400 MHz,CDCl₃) δ 5.86 (s, 3H), 3.77 (s, 6H), 3.52 (m, 2H), 3.44 (t, 2H), 2.66(m, 2H), 2.53 (m, 2H), 2.36 (s, 3H), 1.98 (m, 2H); ¹³C NMR (CDCl₃) δ27.4, 45.3, 48.0, 48.3, 54.8, 55.6, 57.7, 87.4, 90.6, 150.7, 161.3; MS(+) 251.2; HPLC: condition A, RT=4.85 min, 98% homogeneity.

Preparative Example 10: 1-(2-pyridyl)-homopiperazine

At 100° C., homopiperazine (3.82 g, 38.1 mmol) was allowed to react with2-bromopyridine (2 g, 12.7 mmol) using catalytic amount of Pd₂(dba)₃(116 mg, 0.127 mmol) and BINAP (237 mg, 0.381 mmol), NaOtBu (1.71 g,17.78 mmol) with without solvent (neat). The reaction mixture wasstirred under reflux for 1 h30. After cooling, the mixture was taken inDCM and H₂O. The layers were separated and the aqueous layer wasextracted 3 times with DCM. The organic layers were combined, dried(Na₂SO₄) and concentrated to a residue which was purified by columnchromatography on silica gel (9.9/90/0.1 to 14.9/85/0.1 MeOH/DCM/NH₄OH).Evaporation of the collected fractions yielded the title compound as abrown oil (1.23 g, 55% yield). The coupling was repeated with 3 g (19mmol) of 2-bromopyridine to afford after purification 1.46 g of product(43% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.12 (m, 1H), 7.40 (m, 1H), 6.48(m, 2H), 3.70 (m, 4H), 3.01 (m, 2H), 2.82 (m, 2H), 1.86 (m, 2H), 1.69(s, 1H); MS (+) 178.0; HPLC: condition A, RT=1.50 min, 100% homogeneity.

Preparative Example 11: 4-Ethyl-1-(2-pyridyl)-homopiperazine

To a solution of 1-(2-pyridyl)-homopiperazine (2.43 g, 13.73 mmol) int-BuOH (14 mL) was added iodoethane (2.35 g, 15.1 mmol) and Na₂CO₃ (2.91g, 27.46 mmol). The reaction mixture was stirred under reflux for 24 h.After cooling, the mixture was taken in EtOAc and a saturated solutionof NaHCO₃. The layers were separated and the organic layer was washed 4times with a saturated solution of NaHCO₃ and once with brine, dried(Na₂SO₄) and concentrated to a residue which was purified by columnchromatography on silica gel (4.9/95/0.1 to 9.9/90/0.1 MeOH/DCM/NH₄OH).Evaporation of the collected fractions yielded the title compound as abrown oil (1.97 g, 70% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.04 (m, 1H),7.30 (m, 1H), 6.37 (m, 2H), 3.69 (m, 2H), 3.50 (m, 2H), 2.64 (m, 2H),2.45 (m, 4H), 1.88 (m, 2H), 0.96 (t, 3H); ¹³C NMR (CDCl₃) δ 11.9, 27.2,45.9, 46.0, 51.3, 54.1, 54.9, 104.9, 110.8, 136.7, 147.4, 157.8; MS (+)206.0; HPLC: condition A, RT=1.70 min, >99% homogeneity.

RP-HPLC Conditions (Examples)

HPLC analysis were performed on a Waters C18 reversed-phase analyticalcolumn (5 μm, Atlantis, 100×3.9 mm) using a flow rate of 1 mL/min and agradient of 0% to 95% A/B over 15 min (condition A) or on a PhenomenexCN reversed-phase analytical column (5 μm, Luna CN, 150×4.6 mm) with aflow rate of 0.5 mL/min and a gradient of 15% to 95% A/B over 30 min(condition B), where A=CH₃CN+0.1% formic acid+0.1% triethylamine andB=0.1% aqueous formic acid+0.1% triethylamine.

Example 1: 1-Methyl-1-propyl-4-phenyl-homopiperazinium Iodide (ASM-008)

To a solution of 1-phenyl-4-methylhomopiperazine (1.9 g, 10 mmol) inacetone (60 mL) was added 1-propyliodide (8.5 g, 50 mmol). The mixturewas heated to reflux for 15 h, after which it became dark orange. Aftercooling, and evaporation of the solvent to give an orange oil which waspurified by passing through a pad of silica gel and eluted using amixture of 5% MeOH in DCM to give 2.307 g (89% yield) of the titlecompound as a yellow gum: Exact Mass Calcd for C₁₅H₂₅N₂ ⁺[M⁺] 233.2012,found 233.2011.

1,1-dimethyl-4-phenylhomopiperazinium iodide (ASM-002) was prepared in asimilar manner using methyl iodide (10 eq.) and stirring at rt for 25hours Yielding 66% of the compound.

Melting point: 158-160. ¹H NMR DMSO-d6 (ppm): (q, 2H) 7.18, (q, 2H)6.74, (t, 1H) 6.64, (br s, 2H, 3.74), (m, 2H) 3.52, (m, 2H) 3.44, (t,2H) 3.40, (s, 6H) 3.17, (bs s, 2H) 2.21. ¹³C NMR DMSO-d6: 149, 129, 117,112, 66, 65, 53, 47, 43, 22.

Example 2: 1,1-Diethyl-4-phenyl-homopiperazinium Iodide (ASM-009)

To a solution of 1-phenyl-4-ethylhomopiperazine (14.87 g, 73 mmol) inacetone (75 mL) was added ethyliodide (22.74 g, 145.8 mmol). The mixturewas heated to reflux for 22 h, cooled down to room temperature, and theresulting white solid was filtered under vacuum to afford 23.5 g (89%yield) of the title compound: mp=190.5-192.5° C.; ¹H NMR D₂O (ppm): 7.31(dd, 2H), 6.87 (m, 3H), 3.74 (br s, 2H), 3.52 (m, 4H), 3.34 (m, 6H),2.20 (br s, 2H), 1.26 (t, 6H); ¹³C NMR D₂O (ppm): 147.6, 129.4, 118.1,113.1, 60.1, 59.6, 54.6, 46.5, 42.9, 21.5, 6.6; MS ES (+): (M−I⁻)=233.2;100% homogeneity (RT=13.94 min) by LC-MS using CN column with ACN-H₂O(0.1% formic acid) as eluent and UV detection at 240 nm.

Example 3: 1-Ethyl-1-n-propyl-4-phenyl-homopiperazinium Iodide (ASM-010)

To a solution of 1-phenyl-4-ethylhomopiperazine (1.6 g, 7.8 mmol) inacetone (50 mL) was added n-propyliodide (6.8 g, 40 mmol). The mixturewas heated to reflux for 16 h, then allowed to cool to room temperature,and the solvent evaporated to dryness. The orange oily residue waspurified by flash chromatography over silica gel using a gradient of0-7% MeOH in DCM to afford 2.61 g (89% yield) of the title compound as ayellow thick oil: Exact Mass Calcd for C₁₆H₂₇N₂ ⁺[M⁺] 247.2168, found247.2173.

Example 4: 1,1-Dimethyl-4-(2-pyridyl)-homopiperazinium Iodide (ASM-016)

To a solution of 1-methyl-4-(2-pyridyl)-homopiperazine (1.1 g, 5.76mmol) in a 1:1 mixture of acetone/Et₂O (20 mL) was added methyliodide(0.82 g, 5.8 mmol). After stirring at room temperature for 48 h, thesolid formed was filtered, washed with 50 mL Et₂O and then dried 3 hunder vacuum to afford 1.87 g (98% yield) of the title compound as abeige solid: LC-UV-MS analysis: 100% homogeneity (RT=13.49 min) using UVdetection at 240 nm and CN column with ACN-H₂O (0.1% formic acid) asgradient eluent; Exact Mass Calcd for C₁₂H₂₀N₃ ⁺[M⁺] 206.16517, found206.16509; ¹H NMR DMSO-d₆ (ppm): 8.10 (d, 1H), 7.55 (m, 1H), 6.69 (d,1H), 6.63 (m, 1H), 4.03 (br s, 2H), 3.53 (m, 6H), 3.18 (s, 6H), 2.24 (brs, 2H); ¹³C NMR DMSO-d₆ (ppm): 22.1, 39.2, 45.7, 52.5, 64.8, 66.0,106.5, 112.9, 138.2, 147.9, 157.9.

Example 4: 1-Ethyl-1-methyl-4-(2-pyridyl)-homopiperazinium Iodide(ASM-017)

To a solution of 1-methyl-4-(2-pyridyl)-homopiperazine (1.1 g, 5.76mmol) in a 1:1 mixture of acetone/Et₂O (20 mL) was added ethyliodide(3.6 g, 23 mmol). The mixture was stirred at room temperature for 48 h,diluted with 20 mL of Et₂O and the solid was filtered, washed with Et₂O(30 mL), dried under vacuum to afford 1.89 g (95% yield) of the titlecompound as a beige powder: LC-UV-MS analysis: 100% homogeneity(RT=13.60 min) using UV detection at 240 nm and CN column with ACN-H₂O(0.1% formic acid) as gradient eluent; Exact Mass Calcd for C₁₃H₂₂N₃⁺[M⁺] 220.18062, found 220.17991; ¹H NMR DMSO-d₆ (ppm): 8.10 (d, 1H),7.55 (m, 1H), 6.69 (d, 1H), 6.63 (m, 1H), 4.03 (m, 2H), 3.53 (m, 8H),3.18 (s, 3H), 2.24 (m, 2H), 1.25 (t, 3H); ¹³C NMR DMSO-d₆ (ppm): 8.3,21.9, 39.0, 45.6, 48.2, 59.3, 62.3, 63.9, 106.5, 112.9, 138.2, 147.9,157.9.

Example 5: 1-Methyl-1-propyl-4-(2-pyridyl)-homopiperazinium Iodide(ASM-018)

To a solution of 1-methyl-4-(2-pyridyl)-homopiperazine (1.1 g, 5.76mmol) in a 1:1 mixture of acetone/Et₂O (20 mL) was added propyliodide(3.9 g, 23 mmol). The mixture was stirred at room temperature for 72 h,diluted with 25 mL of Et₂O and the mixture was stirred for a further 24h while a with solid was formed. Further dilution with Et₂O (25 mL),filtration and drying under vacuum afforded 1.69 g (81% yield) of thetitle compound as a beige solid: LC-UV-MS analysis: 100% homogeneity(RT=13.92 min) using UV detection at 240 nm and CN column with ACN-H₂O(0.1% formic acid) as gradient eluent; Exact Mass Calcd for C₁₄H₂₄N₃⁺[M⁺] 234.19647, found 234.19618; ¹H NMR DMSO-d₆ (ppm): 8.10 (d, 1H),7.55 (m, 1H), 6.69 (d, 1H), 6.63 (m, 1H), 4.03 (br s, 2H), 3.50 (m, 8H),3.18 (s, 3H), 2.24 (br s, 2H), 1.70 (m, 2H), 0.90 (t, 3H); ¹³C NMRDMSO-d₆ (ppm): 11.0, 15.8, 21.9, 39.0, 45.6, 49.1, 62.8, 64.5, 64.9,106.5, 112.9, 138.2, 147.9, 157.9.

Example 6: 1,1-Diethyl-4-phenyl-homopiperazinium Bromide (ASM-021)

The resin Amberlite® IRA-400(CI) (100 mL) was treated with 2N KBr (250mL), and then washed with 200 mL H₂O. The compound ASM-009 (FG1-62,2.286 g, 6.35 mmol) was dissolved in H₂O (50 mL) by heating slightly andput down on the resin. The product was eluted with water (500 mL) andthe solvent was evaporated. The residue was analyzed by MS and a signalat m/e 127 was present. So, the resin was retreated with 2N KBr and theresidue dissolved in 100 mL H₂O. By MS, always one signal at m/e 127.The resin was washed with deionized water and treated with 2N HBr (500mL), washed with deionized water (350 mL), the residue dissolved indeionized water (100 mL) was passed through the resin. But by MS, 12.5%of I⁻ were still present. So, a new resin was used: Amberlite®IRA-410(CI) (100 mL), treated with 2N HBr (2×250 mL), washed withdeionized water (250 mL). The residue was dissolved in deionized water(50 mL) and eluted from the resin with deionized water. Afterevaporation of the water, an oil was obtained and coevaporated withEt₂O/acetone to afford the title compound as a solid (1.616 g). Thecompound was dried by heating at 40° C. under vacuum for 24 h, buttraces of acetone (5%) were detected by ¹H NMR at 2.22 ppm. The productwas analyzed by ES negative ion mode MS and signals at m/e 125 and 127were present. But, these were adducts Br⁻+HCO₂H (79+46=125; 81+46=127),because when acetic acid was used instead of formic acid, just twosignals were present at m/e 79 and 81: ¹H NMR D₂O (ppm): 7.38 (dd, 2H),6.97 (m, 3H), 3.81 (br s, 2H), 3.56 (m, 4H), 3.41 (m, 6H), 2.24 (br s,2H), 1.29 (t, 6H); MS ES (+): (M−Br⁻)=233.2; 100% homogeneity (RT=14.21min) by LC-MS using CN column with ACN-H₂O (0.1% formic acid) as eluentand UV detection at 240 nm.

Example 7: 1,1-Diethyl-4-phenyl-homopiperazinium Chloride (ASM-022)

The resin Amberlite® IRA-410(CI) (100 mL) was treated with 2N HCl (250mL), washed with 200 mL of distilled water. The compound ASM-009(FG1-62, 2.84 g, 7.89 mmol) was dissolved in H₂O (60 mL) by heatingslightly and put down on the column of resin. The product was elutedwith water (500 mL) and the solvent was evaporated. The residue wastriturated and coevaporated with Et₂O/acetone to afford the titlecompound as a solid (1.755 g). The compound was dried by heating at 40°C. under vacuum for 24 h, but traces of acetone (1%) were detected by ¹HNMR at 2.22 ppm: mp=163.3-164.6° C.; ¹H NMR D₂O (ppm): 7.34 (dd, 2H),6.91 (m, 3H), 3.79 (br s, 2H), 3.56 (m, 4H), 3.46 (m, 6H), 2.24 (br s,2H), 1.28 (t, 6H). By negative ES mode MS, Cl⁻ formed adducts withformic acid: m/e 35+46=81 and m/e 37+46=83; MS ES (+): (M−Cl⁻)=233.2;100% homogeneity (RT=14.12 min) by LC-MS using CN column with ACN-H₂O(0.1% formic acid) as eluent and UV detection at 240 nm.

Example 8: 1,1-Diethyl-4-phenyl-homopiperazinium Acetate (ASM-023)

The resin Amberlite® IRA-410(CI) (100 mL) was treated with 2N HBr (250mL), washed with 250 mL of distilled water, treated with 2N NaOH (50 mL)in an ultrasonic bath for 10 minutes, washed with 2N NaOH (200 mL),treated with 2N AcOH (50 mL) in an ultrasonic bath for 20 minutes,washed with 2N AcOH (200 mL), and finally washed with water (250 mL).Compound ASM-009 (1.68 g, 4.67 mmol) was dissolved in H₂O (60 mL) byheating slightly and eluted through the column of resin. By ES negativeion mode MS, the two signals corresponding to Br⁻ were present. Then theresin was retreated: 2N NaOH (100 mL) in an ultrasonic bath for 15minutes, washed with 2 N NaOH (300 mL), washed with H₂O (250 mL),treated with 2N AcOH (100 mL) in an ultrasonic bath for 25 minutes,washed with 2N AcOH (100 mL) and H₂O (250 mL). The residue was dissolvedin water (30 mL) and eluted through the column of resin with water.After evaporation, the title compound was obtained as an oil (1.40 g):ES negative mode MS: no signals for Br⁻ were detected; ¹H NMR D₂O (ppm):7.21 (dd, 2H), 6.7 (m, 3H), 3.50 (br s, 2H), 3.30 (m, 4H), 3.15 (m, 6H),2.01 (br s, 2H), 1.83 (s, 3H), 1.13 (t, 6H); MS ES (+): (M−AcO⁻)=233.2;100% homogeneity (RT=11.95 min) by LC-MS using CN column with ACN-H₂O(0.1% formic acid) as eluent and UV detection at 240 nm.

Example 9: 1,1-Diethyl-4-phenyl-homopiperazinium Tosylate (ASM-024)

A 5 L three-necked flask, equipped with a mechanical stirrer and acondenser, is charged with 189.55 g (0.93 mol) of1-phenyl-4-ethyl-homopiperazine and then 1 L of acetone. Ethylp-toluenesulfonate (371.6 g, 1.86 mol, 2 equiv) plus 200 mL of acetonefor wash then added and the mixture was heated gently to refluxtemperature. After 4 h, crystals had started to form and 350 ml ofacetone were added to facilitate the stirring. After 24 h, HPLC analysisindicated that there was some starting material left. Consequently, 1additional equivalent of TsOEt was added (186 g) and the mixture wasfurther heated to reflux. After a total of 94 h, HPLC analysis indicatedthat the reaction had not progress much further and therefore, theheating was stopped and after 1 h, t-butyl methyl ether (1 L) was added.The mixture was stirred 15 min, then the crystals were filtered andwashed with 5 portions of 500 mL of t-butyl methyl ether. The fine whiteneedles were dried at room temperature under vacuum for 24 hours toafford 336.86 g (90% yield) of the title compound: mp 167.5°−168.8° C.;LC-UV-MS analysis: 100% homogeneity (RT=13.4 min) using UV detection at240 nm and CN column with ACN-H₂O (0.1% formic acid) as gradient eluent;ES (+) m/z 233.2 (M−TsO⁻); ES (−) m/z 171 (TsO⁻). ¹H NMR (D₂O) δ 7.74(d, 2H), 7.40 (m, 4H), 6.93 (m, 3H), 3.75 (br s, 2H), 3.54 (m, 4H), 3.37(m, 6H), 2.42 (s, 3H), 2.23 (br s, 2H), 1.32 (t, 6H); ¹³C NMR (D₂O) δ6.7, 20.3, 21.5, 42.6, 46.2, 54.5, 59.5, 60.3, 112.8, 117.6, 125.2,129.0, 129.4, 140.4, 141.2, 147.9. The crude product ASM-024 (222.11 g)was dissolved in hot CH₂Cl₂ (750 mL). Then, tBuOMe (160 mL) was addedslowly in order to create a mild milky vein and until the appearance ofthe first crystal, and the mixture was left at room temperature for 3 h.Then, the white solid was filtered, washed with tBuOMe (500 mL) anddried under vacuum. For the second and third recrystallization, the sameprocedure was used with CH₂Cl₂ (750 mL)/tBuOMe (110 mL) and CH₂Cl₂ (720mL)/tBuOMe (120 mL) respectively, to afford 211.7 g of ASM-024 (99%recovery).

Example 10: 1,1-Diethyl-4-phenyl-homopiperazinium Mesylate (ASM-025)

The resin Amberlite® IRA-400(CI) (150-170 mL) was treated with 2N HCl(250 mL), and then washed with 250 mL of distilled water. Then the resinwas washed with 2N NaOH (250 mL), treated with 2N NaOH (50 mL) in anultrasonic bath for 20 minutes, washed with 2N NaOH (200 mL). It waswashed with a 2N solution of methane sulfonic acid (250 mL), treatedwith this 2N solution of methane sulfonic acid (50 mL) in an ultrasonicbath for 30 minutes and washed again with this 2N of methane sulfonicacid (200 mL). Finally the resin washed with water (250 mL) and thecompound ASM-009 (FG1-60, 2.02 g, 5.61 mmol) was dissolved in H₂O (50mL) by heating slightly and put down on the resin. The product waseluted with water (500 mL) and the solvent was evaporated andcoevaporated with EtOH (3×) to afford the title compound as a whitesolid (1.707 g): mp=92.8-94.3° C.; ¹H NMR D₂O (ppm): 7.34 (m, 2H), 6.90(m, 3H), 3.78 (br s, 2H), 3.55 (m, 4H), 3.39 (m, 6H), 2.78 (s, 3H), 2.31(br s, 2H), 1.28 (t, 6H); MS ES (+): (M−MsO⁻)=233.2; 100% homogeneity(RT=13.66 min) by LC-MS using CN column with ACN-H₂O (0.1% formic acid)as eluent and UV detection at 240 nm; MS ES (−): MsO⁻=95.2.

Example 11: 1,1-Diethyl-4-phenyl-homopiperazinium Besylate (ASM-033)

To a solution of 1-phenyl-4-ethyl-homopiperazine (6 g, 29.41 mmol) inacetone (30 mL) was added ethyl benzenesulfonate (10.94 g, 58.82 mmol).The mixture was heated gently to reflux temperature for 25 h. Themixture was cooled to room temperature and filtered. The white solid wasdried at room temperature under vacuum to afford 10.58 g (92% yield) ofthe title compound: LC-UV-MS analysis: 100% homogeneity (RT=10.45 min)using UV detection at 240 nm and CN column with ACN-H₂O (0.1% formicacid) as gradient eluent; ES (+) m/z 233.2 (M−C₆H₅SO₃); ES (−) m/z 157(C₆H₅SO₃); ¹H NMR D₂O (ppm): 7.79 (dd, 2H), 7.53 (m, 3H), 7.33 (m, 2H),6.87 (m, 3H), 3.74 (br s, 2H), 3.51 (m, 4H), 3.35 (m, 6H), 2.20 (brs,2H), 1.26 (t, 6H).

Example 12: 1,1-Diethyl-4-(2-pyridyl)-homopiperazinium Tosylate(ASM-037)

To a solution of 1-ethyl-4-(2-pyridyl)homopiperazine (1.99 g, 9.7 mmol)in acetone (15 mL) was added ethyl p-toluenesulfonate (3.9 g, 19.4mmol). The mixture was first stirred at room temperature. After 18 h,the reaction was not complete, then the mixture was heated gently toreflux temperature for 7 h. There was some starting material left, ethylp-toluenesulfonate (3.9 g, 19.4 mmol) was added and the reaction wasstirred for 4 additional days. The mixture was cooled to roomtemperature and filtered. The white solid was dried at room temperatureunder vacuum to afford 2.46 g (63% yield) of the title compound:LC-UV-MS analysis: 100% homogeneity (RT=13.70 min) using UV detection at240 nm and CN column with ACN-H₂O (0.1% formic acid) as gradient eluent;ES (+) m/z 234.2 (M−TsO⁺); ES (−) m/z 171 (TsO⁺); ¹H NMR D₂O (ppm): 8.03(d, 1H), 7.63 (d, 3H), 7.29 (d, 2H), 6.73 (m, 2H), 3.87 (br s, 2H), 3.60(t, 2H), 3.50 (m, 2H), 3.32 (m, 6H), 2.32 (s, 3H), 2.20 (brs, 2H), 1.26(t, 6H).

Example 13: 1,1-Diethyl-4-(4-chlorophenyl)-homopiperazinium Tosylate(ASM-048)

To a solution of 1-ethyl-4-(4-chlorophenyl)homopiperazine (1.16 g, 4.86mmol) in acetone (6 mL) was added ethyl p-toluenesulfonate (1.95 g, 9.72mmol). The mixture was heated gently to reflux temperature for 17 h. Themixture was cooled to room temperature, then t-butyl methyl ether (10mL) was added. The mixture was stirred 15 min, then filtered and washedwith 2 portions of 10 mL of t-butyl methyl ether. The white solid wasdried at room temperature under vacuum to afford 2.42 g of the titlecompound: LC-UV-MS analysis: 73% A, 27% B (LC-UV), 50% A, 50% B (MS),RT=10.62 min (A), 12.05 min (B) using UV detection at 240 nm and CNcolumn with ACN-H₂O (0.1% formic acid) as gradient eluent; ES (+) A m/z267.2, B m/z 148.2 (M−TsO⁺); ES (−) m/z 171 (TsO⁺).

Example 14: 1,1-Diethyl-4-(4-fluorophenyl)-homopiperazinium Tosylate(ASM-049)

To a solution of 1-ethyl-4-(4-fluorophenyl)homopiperazine (2.27 g, 10.24mmol) in acetone (12 mL) was added ethyl p-toluenesulfonate (4.1 g, 20.5mmol). The mixture was heated gently to reflux temperature for 24 h. Themixture was cooled to room temperature, then t-butyl methyl ether (10mL) was added. The mixture was stirred 15 min, then filtered and washedwith 2 portions of 10 mL of t-butyl methyl ether. The white solid wasdried at room temperature under vacuum to afford 3.50 g (81% yield) ofthe title compound: LC-UV-MS analysis: 100% homogeneity (RT=10.43 min)using UV detection at 240 nm and CN column with ACN-H₂O (0.1% formicacid) as gradient eluent; Exact Mass calcd for C₁₅H₂₄N₂F (M⁺) 251.1915,found 251.1918, C₇H₇SO₃ (M⁻) 171.0117, found 171.0121; mp=169.4-170.5°C.; ¹H NMR D₂O (ppm): 7.61 (d, 2H), 7.28 (d, 2H), 7.01 (t, 2H), 6.80 (m,2H), 3.63 (br s, 2H), 3.46 (m, 2H), 3.33 (m, 8H), 2.31 (s, 3H), 2.13 (brs, 2H), 1.22 (t, 6H); ¹³C NMR D₂O (ppm): 7.7, 21.2, 22.6, 44.7, 48.5,55.7, 60.6, 61.5, 115.7, 115.8, 116.4, 116.7, 126.2, 130.1, 141.0,142.6, 145.8, 145.9, 155.2, 158.3.

Example 15: 1-Ethyl-1-Methyl-4-(phenyl)-homopiperazinium Tosylate(ASM-055)

To a solution of 1-methyl-4-(phenyl)-homopiperazine (2.13 g, 11.21 mmol)in acetone (14 mL) was added ethyl p-toluenesulfonate (4.5 g, 22.42mmol). The mixture was heated gently to reflux temperature for 22 h. Themixture was cooled to room temperature, then t-butyl methyl ether (10mL) was added. The mixture was stirred 15 min, then filtered and washedwith 2 portions of 10 mL of t-butyl methyl ether. The white solid wasrecrystallized twice in dichloromethane/t-butyl methyl ether (40 mL/5mL), then dried at room temperature under vacuum to afford 3.73 g (85%yield) of the title compound: LC-UV-MS analysis: 100% homogeneity(RT=8.20 min) using UV detection at 240 nm and CN column with ACN-H₂O(0.1% formic acid) as gradient eluent; mp=167.5-168.7° C.; ES (+) m/z219.2 (M−TSO⁻); ES (−) m/z 171 (TsO⁻); ¹H NMR D₂O (ppm): 7.64 (d, 2H),7.29 (m, 4H), 6.83 (m, 3H), 3.64 (br s, 2H), 3.29 (m, 8H), 2.94 (s, 3H),2.32 (s, 3H), 2.13 (br s, 2H), 1.27 (t, 3H); ¹³C NMR D₂O (ppm): 6.9,20.0, 21.6, 42.8, 46.2, 48.0, 59.7, 61.9, 62.3, 112.8, 117.9, 124.9,129.0, 129.4, 139.2, 141.9, 147.7.

Example 16: 1,1-Dimethyl-4-(4-methoxyphenyl)-homopiperazinium Tosylate(ASM-057)

To a solution of 4-methyl-1-(4-methoxyphenyl)-homopiperazine (675 mg,3.07 mmol) in acetone (20 mL) was added methyltosylate (1.143 g, 6.14mmol). The reaction mixture was stirred at room temperature for 16 h. Toprecipitate the salt, Et₂O (100 mL) was added to the mixture. Afterfiltration and washing with Et₂O, the title compound was obtained as awhite solid (1.101 g, 88%): mp 174.0-174.5° C.; LC-UV-MS analysis: >99%homogeneity (RT=10.1 min) using UV detection at 240 nm and CN columnwith ACN-H₂O (0.1% formic acid) as gradient eluent; ¹H NMR (400 MHz,D₂O) δ 7.66 (d, 2H), 7.31 (d, 2H), 6.94 (d, 2H), 6.86 (d, 2H), 3.76 (s,3H), 3.63 (m, 2H), 3.57 (m, 2H), 3.49 (m, 2H), 3.42 (m, 2H), 3.12 (s,6H), 2.34 (s, 3H), 2.19 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 20.0, 22.0,44.4, 47.8, 52.6, 52.7, 55.4, 64.7, 114.8, 115.4, 124.9, 129.0, 139.0,142.0, 142.6, 151.5; ES (−) m/z (p-TSO⁻)=171.0; Exact Mass Calcd fora₄H₂₃N₂O⁺[M+H]⁺ 235.1805, found 235.1796.

Example 17: 1,1-Dimethyl-4-(4-trifluoromethylphenyl)-homopiperaziniumTosylate (ASM-058)

To a solution of 4-methyl-1-(4-trifluoromethylphenyl)-homopiperazine(593 mg, 2.30 mmol) in acetone (12 mL) was added methyltosylate (857 mg,4.60 mmol). The reaction mixture was stirred at room temperature for 16h. To precipitate the salt, Et₂O (100 mL) was added to the mixture.After filtration and washing with Et₂O, the title compound was obtainedas a white solid (946 mg, 93%): mp 212.6-213.4° C.; LC-UV-MSanalysis: >99% homogeneity (RT=10.0 min) using UV detection at 240 nmand CN column with ACN-H₂O (0.1% formic acid) as gradient eluent; ¹H NMR(400 MHz, MeOD) δ 7.60 (d, 2H), 7.39 (d, 2H), 7.12 (d, 2H), 6.80 (d,2H), 3.75 (m, 2H), 3.57-3.44 (m, 6H), 3.12 (s, 6H), 2.23 (m, 5H); ¹³CNMR (100 MHz, MeOD) δ 21.4, 23.8, 43.7, 47.7, 53.5, 66.4, 66.8, 113.1,119.1, 120.2, 127.1, 127.8, 130.0, 141.8, 143.8, 152.3; ES (−) m/z(p-TSO)=171.0; Exact Mass Calcd for C₁₄H₂₀N₃F₃ ⁺[M⁺] 273.1573, found273.1577.

Example 18: 1,1-Dimethyl-4-(4-cyanophenyl)homopiperazinium Tosylate(ASM-064)

To a solution of 4-methyl-1-(4-cyanophenyl)-homopiperazine (915 mg, 4.25mmol) in a mixture of Et₂O/acetone (20 mL/10 mL) was addedmethyltosylate (1.581 g, 8.50 mmol). The reaction mixture was stirred atroom temperature for 24 h. To precipitate the salt, Et₂O (150 mL) wasadded to the mixture. After filtration and washing with Et₂O, the titlecompound was obtained as a beige solid (1.300 mg, 71%): mp 202.1-203.2°C.; LC-UV-MS analysis: >99% homogeneity (RT=9.1 min) using UV detectionat 240 nm and CN column with ACN-H₂O (0.1% formic acid) as gradienteluent; ¹H NMR (400 MHz, D₂O) δ 7.63 (d, 2H), 7.45 (d, 2H), 7.20 (d,2H), 6.70 (d, 2H), 3.69 (m, 2H), 3.56 (m, 2H), 3.45 (m, 4H), 3.13 (s,6H), 2.25 (m, 5H); ¹³C NMR (100 MHz, D₂O) δ 20.1, 21.6, 41.7, 45.5,52.2, 64.4, 64.6, 96.7, 111.5, 120.9, 124.7, 129.0, 133.5, 139.7, 141.5,150.9; ES (−) m/z (p-TsO⁺)=171.0; Exact Mass Calcd for C₁₄H₂₀N₃ ⁺[M⁺]230.1652, found 230.1648.

Example 19: 4-Dimethyl-1-(napht-1-yl)-homopiperazinium Tosylate(ASM-067)

To a solution of 4-methyl-1-(napth-1-yl)-homopiperazine (1.17 g, 4.87mmol) in Et₂O (7 mL) and acetone (2 mL) was added methyltosylate (1.82g, 9.75 mmol). The reaction mixture was stirred at room temperature for17 h, filtered and washed with Et₂O to obtain the title compound as awhite solid (2.03 g, 98%): mp 177.6-178.8° C.; LC-UV-MS analysis: 100%homogeneity (RT=10.35 min) using UV detection at 240 nm and CN columnwith ACN-H₂O (0.1% formic acid) as gradient eluent; ES (+) m/z 255.2(M−TsO⁻); ES (−) m/z 171 (TsO⁻). ¹H NMR (400 MHz, DMSO) δ 8.16 (d, 1H),7.91 (d, 1H), 7.65 (d, 1H), 7.50 (m, 5H), 7.23 (d, 1H), 7.10 (d, 2H),3.82 (m, 2H), 3.76 (m, 2H), 3.52 (br m, 2H), 3.24 (m, 8H), 2.25 (m, 5H);¹³C NMR (100 MHz, D₂O) δ 20.9, 22.9, 48.5, 53.6, 55.4, 64.7, 66.4,117.0, 123.5, 123.9, 125.6, 125.8, 126.0, 126.1, 128.2, 128.4, 128.6,134.4, 137.7, 145.8, 149.8; Exact Mass Calcd for [M⁺] C₁₇H₂₃N₂255.18558, found 255.1550.

Example 20: 4-Dimethyl-1-(3-methylphenyl)-homopiperazinium Tosylate(ASM-068)

To a solution of 4-methyl-1-(3-methylphenyl)-homopiperazine (1.12 g,5.52 mmol) in Et₂O (7 mL) and acetone (2 mL) was added methyltosylate(2.05 g, 11.04 mmol). The reaction mixture was stirred at roomtemperature for 21 h, filtered and washed with Et₂O to obtain the titlecompound as a white solid (1.98 g, 92%): mp 161.6-162.5° C.; LC-UV-MSanalysis: 100% homogeneity (RT=10.10 min) using UV detection at 240 nmand CN column with ACN-H₂O (0.1% formic acid) as gradient eluent; ES (+)m/z 219.2 (M−TsO⁻); ES (−) m/z 171 (TsO⁻). ¹H NMR (400 MHz, D₂O) δ 7.55(d, 2H), 7.20 (d, 2H), 7.12 (t, 1H), 6.56 (m, 3H), 3.58 (br m, 2H), 3.45(m, 2H), 3.38 (m, 4H), 3.00 (s, 6H), 2.25 (s, 3H), 2.18 (s, 3H), 2.10(br m, 2H); ¹³C NMR (100 MHz, D₂O) δ 20.0, 20.4, 22.0, 42.9, 46.0, 52.2,52.3, 64.6, 109.7, 113.1, 118.5, 124.9, 129.0, 129.4, 139.1, 139.6,141.9, 147.8; Exact Mass Calcd for [M⁺] C₁₄H₂₃N₂ 219.18558, found219.18553.

Example 21: 4-Dimethyl-1-(napht-2-yl)-homopiperazinium Tosylate(ASM-070)

To a solution of 4-methyl-1-(napht-2-yl)-homopiperazine (1.48 g, 6.17mmol) in Et₂O (8 mL) was added methyltosylate (2.3 g, 12.34 mmol). Thereaction mixture was stirred at room temperature for 18 h, but by MSanalysis, there was some starting material left. Then, methyltosylatewas added (1.15 g, 6.17 mmol) and the mixture was stirred for 5 hadditional, filtered and washed with Et₂O to obtain the title compoundas a white solid (2.53 g, 96%): mp 192.6-193.5° C.; LC-UV-MSanalysis: >99% homogeneity (RT=10.09 min) using UV detection at 240 nmand CN column with ACN-H₂O (0.1% formic acid) as gradient eluent; ES (+)m/z 255.2 (M−TSO⁻); ES (−) m/z 171 (TsO⁻). ¹H NMR (400 MHz, DMSO) δ 7.72(m, 3H), 7.50 (m, 2H), 7.37 (t, 1H), 7.23 (m, 2H), 7.11 (d, 2H), 7.02(s, 1H), 3.87 (br m, 2H), 3.62 (m, 2H), 3.54 (m, 2H), 3.52 (m, 2H), 3.18(s, 6H), 2.28 (m, 5H); ¹³C NMR (100 MHz, DMSO) δ 20.9, 22.0, 42.4, 46.9,52.0, 64.1, 65.1, 105.7, 116.0, 122.1, 125.6, 126.1, 126.3, 126.6,127.3, 128.2, 128.9, 134.8, 137.7, 145.8, 146.4; Exact Mass Calcd for[M⁺] C₁₇H₂₃N₂ 255.18558, found 255.18558.

Example 22: 4-Dimethyl-1-(4-trifluorophenyl)-homopiperazinium Iodide(ASM-071)

To a solution of 4-methyl-1-(4-trifluorobenzyl)-homopiperazine (1.5 g,5.81 mmol) in Et₂O (8 mL) was added iodomethane (1.65 g, 11.63 mmol).The reaction mixture was stirred at room temperature for 24 h, filteredand washed with Et₂O to obtain the title compound as a beige solid (2.32g, 100%): mp 224.3-225.4° C.; LC-UV-MS analysis: 100% homogeneity(RT=4.83 min) using UV detection at 240 nm and CN column with ACN-H₂O(0.1% formic acid) as gradient eluent; ES (+) m/z 273.0 (M−I⁻); ES (−)m/z 127.0 (1). ¹H NMR (400 MHz, DMSO) δ 7.50 (d, 2H), 6.92 (d, 2H), 3.87(br m, 2H), 3.57 (m, 2H), 3.50 (m, 4H), 3.19 (s, 6H), 2.28 (br m, 2H);¹³C NMR (100 MHz, DMSO) δ 21.6, 41.5, 46.7, 52.0, 64.1, 64.8, 111.7,115.9, 116.2, 116.5, 116.9, 123.9, 126.3, 126.4, 126.5, 126.6, 151.0;Exact Mass Calcd for [M⁺] C₁₄H₂₀F₃N₂273.15731, found 273.15862.

Example 23: 4-Diethyl-1-(2-pyridyl)-homopiperazinium Iodide (ASM-072)

To a solution of 4-ethyl-1-(2-pyridyl)-homopiperazine (1.81 g, 8.83mmol) in Acetone (12 mL) was added iodoethane (2.75 g, 17.66 mmol). Thereaction mixture was stirred at room temperature for 24 h, filtered andwashed with Et₂O to obtain the title compound as a white solid (3.03 g,95%): mp 166.3-167.2° C.; LC-UV-MS analysis: >99% homogeneity (RT=10.55min) using UV detection at 240 nm and CN column with ACN-H₂O (0.1%formic acid) as gradient eluent; ES (+) m/z 234.2 (M−I⁻); ES (−) m/z127.0 (I⁻). ¹H NMR (400 MHz, D₂O) δ 7.92 (m, 1H), 7.52 (m, 1H), 6.64 (m,2H), 3.79 (br s, 2H), 3.53 (m, 2H), 3.45 (m, 2H), 3.29 (m, 6H), 2.14 (brs, 2H), 1.92 (t, 6H); ¹³C NMR (100 MHz, D₂O) δ 6.7, 21.3, 40.0, 44.3,54.3, 59.3, 60.6, 107.0, 112.0, 138.7, 146.5, 156.9; Exact Mass Calcdfor [M⁺] C₁₄H₂₄N₃ 234.19647, found 234.19685.

Example 24: 1,1-Diethyl-4-(phenyl-4-hydroxy)-homopiperazinium tosylate(ASM-073)

Step 1

To a solution of benzyl 1-homopiperazine carbon/late (5.27 g, 22.5 mmol)in t-BuOH (30 mL) were added EtI (4.2 g, 27 mmol) and Na₂CO₃ (4.8 g, 45mmol) at 0° C. Then, the mixture was stirred at reflux temperature for2.5 h. The volatile was evaporated and the residue was dissolved in H₂Oand extracted 3 times with Et₂O. The organic layer was washed withbrine, dried under Na₂SO₄, filtered and evaporated to afford crudeproduct 1-(Benzylcarboxy)-4-ethyl-homopiperazine (4.95 g, 84% yield) asa orange oil: (M+H)⁺: 263.6.

Step 2:

To a solution of 1-(benzylcarboxy)-4-ethyl-homopiperazine (4.95 g, 18.89mmol) in EtOAc (110 mL) was added 10% Pd/C (1 g). The mixture wasfilled, vented, and filled 3 times with hydrogen and stirred at roomtemperature under H₂ atmosphere for 17 h. The catalyst was filtered onCelite®, washed with EtOAc, and the filtrate and washings were combinedand evaporated to provide crude product 1-Ethyl-homopiperazine (2 g, 83%yield) as an oil: 1H NMR (400 MHz, CDCl3) δ 2.90 (m, 4H), 2.59 (m, 6H),1.73 (m, 3H), 1.04 (m, 3H).

Step 3:

To a solution of 1-ethyl-homopiperazine (740 mg, 5.78 mmol) in toluene(7 mL) were added 4-benzyloxybromobenzene (2 g, 7.51 mmol), KOtBu (8.7mL, 8.7 mmol), Pd₂(dba)₃ (264.6 mg, 0.289 mmol) and BINAP (540 mg, 0.867mmol). The mixture was stirred at 90° C. for 18 h, then cooled down toroom temperature. The mixture was diluted with EtOAc (50 mL) and H₂O (25mL). After the separation of the layers, the organic phase was washedonce with H₂O (25 mL). The combined aqueous phases were back extractedwith EtOAc (25 mL). The resulting combined organic phases were extractedwith a 2N HCl solution (3×20 mL). The aqueous phases were combined,cooled down with an ice bath and basified up to pH 10 with a 5N NaOHsolution. The resulting aqueous phase was extracted with DCM (3×20 mL),washed with brine (25 mL), dried over Na₂SO₄, filtered, and concentratedunder reduced pressure to afford product1-(phenyl-4-benzyloxy)-4-ethyl-homopiperazine as a brown oil (1.52 g,85% yield): ¹H NMR (400 MHz, CDCl₃) δ: ¹H NMR (400 MHz, CDCl₃) δ 7.41(m, 5H), 6.92 (m, 2H), 6.67 (m, 2H), 5.03 (s, 2H), 3.54 (t, 2H), 3.46(t, 2H), 2.78 (m, 2H), 2.61 (m, 4H), 2.01 (m, 2H), 1.10 (t, 3H); (M+H)⁺:311.2.

Step 4:

To a solution of 1-(phenyl-4-benzyloxy)-4-ethyl-homopiperazine (4.51 g,14.55 mmol) in acetone (20 mL) was added EtOTs (8.73 g, 43.65 mmol). Themixture was stirred at reflux for 41 h, and after cooling down, MTBE (30mL) was added. After stirring 15 min, the precipitate was filtered andwashed with MTBE, dried under vacuum to afford product1,1-Diethyl-4-(phenyl-4-benzyloxy)-homopiperazinium tosylate (6.88 g,93%) as a beige solid: ¹H NMR (400 MHz, CD₃OD) δ 7.72 (d, 2H), 7.36 (m,5H), 7.24 (d, 2H), 6.93 (m, 2H), 6.82 (m, 2H), 5.03 (s, 2H), 3.67 (br s,2H), 3.61 (m, 2H), 3.46 (m, 8H), 2.38 (s, 3H), 2.23 (br s, 2H), 1.33 (t,6H); (M)⁺: 339.3.

Step 5:

To a solution of 1,1-diethyl-4-(phenyl-4-benzyloxy)-homopiperaziniumtosylate (6.87 g, 13.47 mmol) in DCM (90 mL) was added 10% Pd/C (700mg). The mixture was filled, vented, and filled 3 times with hydrogenand stirred at 40° C. under H₂ atmosphere for 21 h. The reaction wasincomplete by MS analysis, the catalyst was filtered on Celite®, washedwith MeOH, and the filtrate and washings were evaporated. The residuewas coevaporated with DCM, and dissolved in DCM (90 mL), 10% Pd/C (700mg) was added, the mixture was filled, vented, and filled 3 times withhydrogen and was heated at 40° C. for an additional 7 h. However, therewas some material left, the catalyst was filtered on Celite®, washedwith MeOH, and the filtrate and washings were evaporated. The residuewas coevaporated with DCM, and dissolved in DCM (90 mL), 10% Pd/C (700mg) was added. The mixture was filled, vented, and filled 3 times withhydrogen and stirred at 40° C. under H₂ atmosphere for an additional 15h. The catalyst was filtered on Celite®, washed with MeOH, and thefiltrate and washings were evaporated. The crude solid was trituratedwith MTBE and filtered, then triturated 3 times with DCM to afford afterdrying under vacuum the title compound as a beige solid (4.25 g, 75%):mp=144.0-145.5° C.; ¹H NMR (400 MHz, CD₃OD) δ 7.72 (d, 2H), 7.24 (d,2H), 6.75 (m, 4H), 3.60 (br s, 4H), 3.45 (m, 8H), 2.38 (s, 3H), 2.20 (brs, 2H), 1.32 (t, 6H); ¹³C NMR (100 MHz, CD₃OD) δ 7.5, 20.7 23.4, 46.0,49.9, 55.7, 60.9, 62.6, 116.4, 117.1, 126.4, 129.3, 141.1, 143.1, 143.7,151.2; HPLC: condition B, RT=5.92 min, 100% homogeneity; ES-MS[p-TsO⁻]=171.0; Exact Mass Calcd for [M⁺] C₁₅H₂₅N₂O 249.19614, found249.19575.

Example 25: Cell Proliferation Assay

In vitro experiments were conducted on four cancer cell lines: the A549adenocarcinoma cell line, the H520 squamous cell carcinoma the H82 smallcell lung carcinoma and Calu 3 lung adenocarcinoma. The A549 tumorcells, were plated at a density of 1.5×10⁴ cells per mL and let toadhere on 96-well microplates in F12K cell culture medium enriched with10% fetal calf serum (FCS). The H520, H82 and Calu 3 tumor cells wereplated at a density of 5×10⁴ cells per mL in RPMI+10% FCS. After 24 h inculture, the medium was changed and cells were exposed to ASM-024 atconcentrations ranging from 10-⁷M to 10⁻³M (FIG. 1). After 3 and 6 daysin culture, cell growth was quantified using the MTT colorimetric assay(M2128, Sigma) at 575-690 nm or the Cell Titer fluorescence assay(G8080, Promega) at 560/590 nm. Tiotropium, a long-acting muscarinicreceptor antagonist and ipratropium, a short-acting muscarinic receptorantagonist were tested as well under the same culture conditions atconcentrations ranging from (10⁻⁹ to 10⁻⁴M) (FIGS. 2 and 3).

The data illustrated in the figures show a dose-dependent decrease incell proliferation on day 3 and day 6 by ASM-024 whereas, except for acytotoxic effect of ipratropium at 1000 μM on A549, no significant cellgrowth inhibition is observed with either tiotropium and ipratropium.

In an another set of experiments, a series of 16 other homopiperaziniumcompounds, in addition to ASM-024, were tested for theiranti-proliferative effect on the four tumor cell lines described aboveafter up to 6 days in culture at concentrations of 10-7M to 10-3M. (FIG.4) Cell growth was quantified using the MTT colorimetric assay for theA-549, H520 and Calu 3 cell lines and the CellTiter Fluorescence assay.for the H82 tumor cell line These are established method of determiningviable cell number in proliferation studies. The preliminary dataindicate an inhibitory effect of cell growth for cells exposed to thehomopiperazinium compounds compared to vehicle treated cells; thiseffect appears to be cell line dependant, as well as concentration andtime-dependant as a more pronounced inhibition is observed at a higherconcentration and after 6 days vs 3 days. in culture. No IC50 could becalculated for Tiotropium

Activity¹ IC₅₀ (μM) Entry Compound N Day 3 Day 6 ASM-002

3 40 ± 15 38 ± 12 ASM-009

3 51 ± 6  48 ± 25 ASM-016

3 90 ± 15 58 ± 22 ASM-017

3 89 ± 13 65 ± 25 ASM-018

3 136 ± 39  82 ± 32 ASM-021

3 45 ± 12 51 ± 39 ASM-022

3 46 ± 7  45 ± 32 ASM-024

10  56 ± 20 59 ± 21 ASM-037

3 146 ± 92  112 ± 96  ASM-048

1 37 15

ASM-049

4 81 ± 74 42 ± 34 ASM-055

4 60 ± 26 28 ± 15 ASM-058

5 22 ± 10 27 ± 26 ASM-068

4 36 ± 17 20 ± 16 ASM-071

3 25 ± 15 18 ± 9  ASM-073

3 119 ± 49  161 ± 82  ¹Activity expressed as the drug concentrationrequired to inhibit cell growth by 50%.

Activity¹ IC₅₀ (μM) Entry Compound N Day 3 Day 6 ASM-002

3 323 ± 60  170 ± 23  ASM-009

3 892 ± 98  396 ± 147 ASM-016

3 415 ± 212 180 ± 92  ASM-017

3 953 ± 39  469 ± 163 ASM-018

3 >1000 536 ± 101 ASM-021

3 >1000 369 ± 171 ASM-022

3  >700 387 ± 178 ASM-024

8 468 ± 287 257 ± 257 ASM-037

3 >1000 634 ± 124 ASM-048

1  322 169

ASM-049

4  >700 288 ± 61  ASM-055

4 622 ± 211 272 ± 65  ASM-058

5 356 ± 143 210 ± 82  ASM-068

4 580 ± 85  233 ± 31  ASM-071

3 523 ± 292 235 ± 179 ASM-073

3 187 ± 28  148 ± 14  ¹Activity expressed as the drug concentrationrequired to inhibit cell growth by 50%.

Activity¹ IC₅₀ (μM) Entry Compound N Day 3 Day 6 ASM-002

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: −15; 3 × 10⁻⁴ M: 31 ASM-009

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: −12; 3 × 10⁻⁴ M: 35 ASM-016

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: −22; 3 × 10⁻⁴ M: −6 ASM-017

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: −26; 3 × 10⁻⁴ M: −6 ASM-018

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 15; 3 × 10⁻⁴ M: 35 ASM-021

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 13; 3 × 10⁻⁴ M: 59 ASM-022

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 23; 3 × 10⁻⁴ M: 55 ASM-024

3 314 ± 205 218 ± 76  ASM-037

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 10; 3 × 10⁻⁴ M: 29 ASM-048

1 246 176

ASM-049

1 331 252 ASM-055

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 36; 3 × 10⁻⁴ M: 66 ASM-058

1 472 299 ASM-068

1 298 179 ASM-073

% Inhibition of Cell Growth at day 3 compared to vehicle-treated cells:10⁻⁴ M: 100; 3 × 10⁻⁴ M: 100 ¹Unless specified otherwise, activity isexpressed as the drug concentration required to inhibit cell growth by50%.

Activity¹ IC₅₀ (μM) Entry Compound N Day 3 Day 6 ASM-024

2 685 ± 320 192 ± 217 ASM-048

1 276  86

ASM-049

1 452 109 ASM-068

1 >1000  52 ¹Activity expressed as the drug concentration required toinhibit cell growth by 50%.

Example 26: In Vitro Anti-Proliferative Properties in Other Cancer CellLines

The effect of ASM-024 on the growth of MCF7, SK-BR-3, MDA-MB-231, BT-474(mammary adenocarcinomas), SK-OV/3 (ovary adenocarcinoma), PC3 (prostateadenocarcinoma) SK-MEL-28 (skin melanoma) and C6 (tumor cell glioma) wasevaluated. Cells were cultured in RPMI medium and incubated withdifferent concentrations of ASM-024. Cell proliferation was measured ondays 3 and 6 by the MTT colorimetric assay. Absorbance at 576-690 nm wasanalyzed with a microplate reader and expressed as optical density (OD)and as a percentage of the value of corresponding untreated cells. TheIC₅₀ was determined as the drug concentration required to inhibit cellgrowth by 50% from a plot of percent cell viability from controluntreated cells versus treated cells.

FIGS. 5a and 5b illustrate the observed results for the MDA-MB-231mammary/breast gland adenocarcinoma assay. Cells were incubated with thedose drug shown and growth measured at the days indicated. In FIG. 5athe data is expressed as optical density (OD) and in FIG. 5b as apercentage of the value of corresponding untreated cells. P<0.05compared to control at same day by Student t-test

IC₅₀ Cell line Cancer Type Day 3 Day 6 MCF7 Mammary/breast gland 470 ±364 209 ± 42 adenocarcinoma SK-BR-3 Mammary/breast cancer >1000 197 ± 67adenocarcinoma MDA-MB-231 Mammary/breast gland >1000 661 ± 115adenocarcinoma BT-474 Breast/duct adenocarcinoma >1000 644 ± 230 SK-OV/3Ovary adenocarcinoma >600 618 ± 125 SK-MEL-28 Skin melanoma >800 259 ±63 C6 Glioma 353 ± 52  186 ± 27 PC3 Prostate adenocarcinoma >700 176 ±29

Example 27: In Vivo Anti-Tumor Properties

For the in vivo antitumor assay, nu/nu mice were inoculatedsubcutaneously in the flank of mice with 2×106 A549 cells in sterile PBSmixed with 50% matrigel. Two days later ASM-024 was administered dailyby subcutaneous (at a site close to the tumor) or intraperitonealdelivery at the dose of 30 mg/kg for up to 50 days. When palpable, tumorlength and width were measured with a caliper and tumor volumecalculated according to the formula Volume=(width2×length)/2.

FIGS. 6a, 6b, 7a and 7b are illustrating the antitumor effect of ASM-024in A549 tumor cell-bearing mice after 51 days. The antitumor effect ofeach delivery mode was evaluated by measuring tumor volume (FIGS. 6a and6b ) and weight (FIGS. 7a and 7b ). Values are presented as themean±standard deviation.

The in vivo antitumor effects of ASM-024, administered by subcutaneousor intraperitoneal delivery, on A549 cells were investigated in atransplanted tumor nude mice model. Tumor growth, as determined by meantumor volumes, was reduced after treatment with ASM-024 at a dose of 30mg/kg during the 51 days of observation, compared to the control mice(FIGS. 6a and 6b ). Mean tumor weight after surgical resection alsoshows a reduction in tumor mass after treatment with ASM-024 compared tovehicle-treated control mice (FIGS. 7a and 7b ).

Example 28: In Vitro Antitumor Activity of ASM-024-Cisplatin andASM-024-Taxol Combination Treatments in A549 and MCF7 Cells

The concomitant effect of ASM-024 and cisplatin or taxol on theproliferation of A549 lung adenocarcinoma and MCF7 breast canceradenocarcinoma cells was assessed in vitro.

Method

A549 cells (3.0×10³ cells/well) were first cultured with 10% FBS in F12Kmedia and MCF7 cells (10×10³ cells/well) with 10% FBS in EMEM media for24 hours to allow cells to adhere to the plate at 37° C. in a humidifiedincubator with 5% CO2. The next day, the media was replaced in mediumcontaining appropriate drug concentrations for two days for anadditional 72 hours. Cell viability was quantified using the MTTcolorimetric assay (M2128, Sigma) at 575-690 nm.

Results

FIG. 8A and FIG. 8B show the percentage of A549 cell growth inhibitionafter 3 days of drug treatment. ASM-024 induced a dose-dependent growthinhibition of cell proliferation, the concomitant addition of increasingconcentrations of Cisplatin (Hospira) or taxol (Paclitaxel, BiolysePharma) to suboptimal doses of ASM-024 potentiated the inhibition of invitro proliferation.

FIG. 9A and FIG. 9B show the percentage of MCF7 cell growth inhibitionafter 3 days of drug treatment. A similar potentiated inhibition de cellproliferation was observed following combination treatments of ASM-024and the chemotherapeutic agents.

These results indicate that concomitant treatment with ASM-024 couldhave clinical applications. ASM-024 may overcome drug resistance toconventional therapeutic doses and thus increase therapeutic efficacyand or allow the administration of lower doses of these drugs thusdecreasing their cytotoxicity.

Example 29: In Vivo Antitumor Activity of ASM-024, Compared to Cisplatinand Taxol Treatments in Xenografts Models of Human Lung and BreastCancers

Method

For the in vivo antitumor assay, A549 human lung carcinoma cells(3.5×106) in 50% F12K medium (Matrigel) were implanted subcutaneously inthe flank of Balb/c nude mice (nu/nu, Charles River) and allowed to growfor 14 days. ASM-024 was administered daily by subcutaneous delivery atthe dose of 45 mg/kg from day 14 to day 38 post cell inoculation. Taxolwas administrated intraperitoneally at the dose of 10 mg/kg on days 21,28 and 35. Cisplatin was administrated intraperioneally at the dose of2.5 mg/kg on days 28 and 35. From day 14 to day49, tumor length andwidth were measured once or twice a week with a caliper and tumor volumecalculated according to the formula Volume=(width2×length)/2. Atsacrifice tumor weight was also assessed.

Results

After 49 days of treatment, a decrease in A549 human carcinoma tumorgrowth is observed in cisplatin, taxol or ASM-024 treated mice (seeFIGS. 10A and 10B). While the invention has been described in connectionwith specific embodiments thereof, it is understood that it is capableof further modifications and that this application is intended to coverany variation, use, or adaptation of the invention following, ingeneral, the principles of the invention and including such departuresfrom the present disclosure that come within known, or customarypractice within the art to which the invention pertains and as may beapplied to the essential features hereinbefore set forth, and as followsin the scope of the appended claims. All references cited herein areincorporated by reference in their entirety.

1. A method for treating cancer comprising administering to a patient inneed thereof an effective amount of a compound having the formula:

wherein R₁ and R₂ are independently alkyl of 1 to 6 carbon atoms orcycloalkyl of 3 to 6 carbon atoms, Xa is CH or N, Ya is hydrogen or asubstituent, each of which is independently selected, n is an integerfrom 1 to 5, J is a counter ion.
 2. The method as defined in claim 1,wherein R₁ and R₂ are independently selected from methyl, ethyl,n-propyl, or i-propyl.
 3. The method as defined in claim 1, wherein Xais CH.
 4. The method as defined in claim 1, wherein Xa is N.
 5. Themethod as defined in claim 1, wherein Ya is hydrogen or independentlyselected from halogen, cyano, hydroxyl, alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 6 carbon atoms, heteroaryl of 6 members and aryl.
 6. Themethod as defined in claim 1, wherein n is 1 or
 2. 7. The method asdefined in claim 1, wherein R1 and R2 are independently selected frommethyl, ethyl, n-propyl, or i-propyl; Xa is N or CH; Ya is hydrogen orindependently selected from halogen, cyano, hydroxyl, alkyl of 1 to 6carbon atoms, alkoxy of 1 to 6 carbon atoms, heteroaryl of 6 members andaryl; n is 1 or 2; wherein J⁻ is fluoride, chloride, bromide, iodide,acetate, sulfate or sulfonate.
 8. The method as defined in claim 1,wherein R1 and R2 are independently selected from methyl, ethyl,n-propyl, or i-propyl; Xa is CH; Ya is hydrogen or independentlyselected from halogen, cyano, hydroxyl, alkyl of 1 to 6 carbon atoms,alkoxy of 1 to 6 carbon atoms, heteroaryl of 6 members and aryl; n is 1;wherein J⁻ is fluoride, chloride, bromide, iodide, acetate, sulfate orsulfonate.
 9. The method as defined in any claim 1, wherein saidcompound has the formula:


10. The method as defined in claim 1, wherein said cancer is acarcinoma, a sarcoma, a melanoma; a lymphoma, a leukemia; a myeloma; ablastoma; a germ cell tumor; a glioma or a CNS cancer(s).
 11. The methodas defined in claim 1, wherein said cancer is a carcinoma.
 12. Themethod as defined in claim 11, wherein said carcinoma is a cancer of thelung.
 13. The method as defined in claim 7, wherein said sulfonate istosylate, mesylate or besylate.
 14. The method as defined in claim 13,wherein said cancer is carcinoma.
 15. The method as defined in claim 14,wherein said carcinoma is a cancer of the lung.
 16. The method asdefined in claim 8, wherein said sulfonate is tosylate, mesylate orbesylate.
 17. The method as defined in claim 16, wherein said cancer iscarcinoma.
 18. The method as defined in claim 17, wherein said carcinomais a cancer of the lung.
 19. The method as defined in claim 9, whereinsaid cancer is carcinoma.
 20. The method as defined in claim 19, whereinsaid carcinoma is a cancer of the lung.